Correlated fluorescence lifetime and spectral measurements in living cells

Studies of proteins' interaction in cells by FRET can take benefit from two important photo-physical properties describing fluorescent proteins: fluorescence emission spectrum and fluorescence lifetime. These properties provide specific and complementary information about the tagged proteins and their environment. However, none of them taken individually can completely quantify the involved fluorophore characteristics due to their multiparametric dependency with molecular environment, experimental conditions, and interpretation complexity. A solution to get a better understanding of the biological process implied at the cellular level is to combine the spectral and temporal fluorescence data acquired simultaneously at every cell region under investigation. We present the SLiM-SPRC160, an original temporal/spectral acquisition system for simultaneous lifetime measurements in 16 spectral channels directly attached to the descanned port of a confocal microscope with two-photon excitation. It features improved light throughput, enabling low-level excitation and minimum invasivity in living cells studies. To guarantee a fairly good level of accuracy and reproducibility in the measurements of fluorescence lifetime and spectra on living cells, we propose a rigorous protocol for running experiments with this new equipment that preserves cell viability. The usefulness of SLiM approach for the precise determination of overlapping fluorophores is illustrated with the study of known solutions of rhodamine. Then, we describe reliable FRET experiments in imaging mode realized in living cells using this protocol. We also demonstrate the benefit of localized fluorescence spectrum-lifetime acquisitions for the dynamic study of fluorescent proteins. proteins.     

Methane calibration burner and C‐factor determination with cone calorimeter – validation of the standardized calibration protocol

The cone calorimeter is one of the major fire tests. In its reference standard ISO 5660‐1, the apparatus (and especially a characteristic of its orifice plate mass flow meter, called C‐Factor) is calibrated using a methane burner set at 5 kW. However, measurements performed in end‐use are in the range 1–10 kW. In addition, standard proposes to check linearity at 1 and 3 kW, which means in the lowest part of the range. This communication establishes the validation of the linearity on the whole range and determines the optimal condition for C‐factor determination. It justifies the technical choices of the standard. Copyright © 2014 John Wiley & Sons, Ltd.     

Poly(L‐lactic acid) with added α‐tocopherol and resveratrol: optical,physical, thermal and mechanical properties

Poly(L ‐lactic acid) (PLLA) films containing various concentrations of two natural antioxidants, α‐tocopherol and resveratrol, were fabricated by a melt compounding and compression molding process. The influence of the antioxidants on the optical properties such as color and UV‐visible light transmission was analyzed. The thermal, mechanical, rheological and physical properties of PLLA films with added antioxidants were assessed. PLLA films with added α‐tocopherol and resveratrol showed a yellowish brown color and the lightness was influenced by the presence of the antioxidants. The glass transition and melting temperatures were significantly reduced with the addition of antioxidants while enhanced thermal stability was observed, which could be a benefit and important for processing and production. PLLA films with added antioxidants were slightly more hydrophobic than neat PLLA. The combination effect of plasticizing and enhancement of the elastic modulus with differing concentrations of two antioxidants played a critical role in the mechanical and thermomechanical properties of PLLA films. The melt viscosity of the PLLA films with added antioxidants was substantially higher than that of neat PLLA. The higher melt viscosity and G′(ω) could be an indication of formation of entanglement between PLLA and the two antioxidants. Copyright © 2012 Society of Chemical Industry     

Development of the Analysis of Fecal Stanols in the Oyster Crassostrea gigas and Identification of Fecal Contamination in Shellfish Harvesting Areas

The objective of this work was to study the effects of washing and purification steps on qualitative and quantitative analysis of fecal stanols in the oyster Crassostrea gigas using either single or a combination of lipid purification steps on silica gel or aminopropyl bonded silica gel (NH₂) or a washing step. Among the three analytical pathways compared, the two including water extraction or NH₂ purification did not lead to higher recoveries and decreased repeatabilities of extractions compared to the single purification on silica gel. This latter led to similar recoveries (ca. 80 %) and repeatabilities (ca. 10 %) for both spiked standards (coprostanol and sitostanol). This analytical pathway has been applied to oysters collected in a harvesting area in Brittany (France) where fecal contaminations are important and allowed to quantify eight stanols in oysters. The relative proportions of fecal stanols of these oysters were combined with principal component analysis in order to investigate the usefulness of their stanol fingerprints to record a fecal contamination and to distinguish its source between human, porcine and bovine contaminations. Oysters non-fecally contaminated by Escherichia coli did not present specific stanol fingerprints while oysters fecally contaminated had a bovine fingerprint, suggesting a contamination of these samples by bovine sources. As a consequence, the method developed here allows the use of stanol fingerprints of oysters as a microbial source tracking tool that can be applied to shellfish harvesting areas subjected to fecal contaminations in order to identify the different sources of contamination and improve watershed management.     

Electroseparation of an antibacterial peptide fraction from snow crab by-products hydrolysate by electrodialysis with ultrafiltration membranes

Recently, a snow crab by-products hydrolysate has demonstrated antibacterial properties due to a peptide with a molecular weight of about 800 Da, but only at high concentration. Consequently, peptide hydrolysate has been fractionated to obtain peptides in a more purified form. The aim of this work was to separate a snow crab by-products hydrolysate by electrodialysis with ultrafiltration membranes (EDUF). EDUF, which allows separation of molecules according to their charges and molecular weights, was used to recover and concentrate the active antibacterial fraction. Two different ultrafiltration membranes (20 and 50 kDa) and two electrical field strengths (2 and 14 V/cm) were used as separation parameters. After EDUF separation, the 300-600 Da peptide molecular weight range was the most recovered with an abundance of 94%. Moreover, fractionation at 14 V/cm with ultrafiltration membranes of 50 kDa allowed the recovery of an anionic fraction which showed antibacterial properties on Escherichia coli ATCC 25922 and Listeria innocua HPB 13.     

Carvacrol and trans-Cinnamaldehyde Reduce Clostridium difficile Toxin Production and Cytotoxicity in Vitro

Clostridium difficile is a nosocomial pathogen that causes a serious toxin-mediated enteric disease in humans. Reducing C. difficile toxin production could significantly minimize its pathogenicity and improve disease outcomes in humans. This study investigated the efficacy of two, food-grade, plant-derived compounds, namely trans-cinnamaldehyde (TC) and carvacrol (CR) in reducing C. difficile toxin production and cytotoxicity in vitro. Three hypervirulent C. difficile isolates were grown with or without the sub-inhibitory concentrations of TC or CR, and the culture supernatant and the bacterial pellet were collected for total toxin quantitation, Vero cell cytotoxicity assay and RT-qPCR analysis of toxin-encoding genes. The effect of CR and TC on a codY mutant and wild type C. difficile was also investigated. Carvacrol and TC substantially reduced C. difficile toxin production and cytotoxicity on Vero cells. The plant compounds also significantly down-regulated toxin production genes. Carvacrol and TC did not inhibit toxin production in the codY mutant of C. difficile, suggesting a potential codY-mediated anti-toxigenic mechanism of the plant compounds. The antitoxigenic concentrations of CR and TC did not inhibit the growth of beneficial gut bacteria. Our results suggest that CR and TC could potentially be used to control C. difficile, and warrant future studies in vivo.     

Toward green dialysis: Focus on water savings

Hemodialysis is one of the most water and energy‐hungry medical procedures, and thus represents a clear opportunity where improvements should be made concerning the consumption and wastage of water. Three levels were investigated on which there are potential savings: the precise adjustment of water production according to specific needs, the reuse of reverse osmosis rejected water, and finally the huge volumes of post‐patient dialysate effluent. The “AURAL” (Association pour l′Utilisation du Rein Artificiel à Lyon), main unit in Lyon, was the site of investigation for this study, which cares for 173 chronic hemodialysis patients. Evaluation of the 3 levels described earlier was undertaken on this particular building, and on the water treatment currently used. Volumes of produced water can be improved by different hydraulic systems or by adjusting the pure water conductivity used for dialysis. Concerning the reject water, reuse for building sanitation became the focus of further attention. The technical feasibility, volume of saved water, and applicable work costs were considered. The results suggest that out of a possible 2834 m³/year of reject water, 1200 m³/year may be reused and return on investment recovered within 5.8 years. Finally, the reprocessing and feasibility of reuse of dialysate effluent were investigated. Initial calculations show that although technical solutions are available, such processing of the wastewater production is not profitable in the short term. Regarding the significant prior authorization and risk management analysis necessary for such a project, this avenue was pursued no further. From the perspective of a “green dialysis,” the reuse of reject water into sanitation is both viable and profitable in our unit, and must be the next step of our project. More widely, improvements can be made by defining a more precise range of pure water conductivity for dialysis and by applying reuse water project to new or to be renovated units.     

On the applicability of the Heliosat-2 method to assess surface solar irradiance in the Intertropical Convergence Zone,French Guiana

Measurements of daily means of surface solar irradiance made at four ground stations in French Guiana are compared to estimates from the HelioClim-3 database produced by the Heliosat-2 method applied to Meteosat satellite images. The bias ranges from 12 W m^?2 (6% of the mean of measurements) to 23 W m^?2 (12%), depending on the stations. The root mean square difference ranges between 23 W m^?2 (11%) and 35 W m^?2 (18%). The correlation coefficient (r) is close to 0.9. Better results are observed during the rainy season than during the dry season. Uncertainties are mainly due to the presence of clouds, large viewing angles of the Meteosat satellite, and limitations of the atmospheric transmittance model under the tropical atmospheric conditions. It is concluded that the Heliosat-2 method provides new knowledge about solar radiation in French Guiana.